Phosphorylation of CREB at Serine 142 and 143 Is Essential for Visual Cortex Plasticity

Viral constructs and functional validation. A, Diagrams of HSV constructs used in this study, as described in Materials and Methods. The different domains of the CREB protein are shown in blue, with the dominant negative mutation indicated. The fluorescent tag (GFP) is in the green box. The yellow arrows are the promoters for each gene (IE 4/5 for CREB, and CMV for GFP). B, Western blots showing that phosphorylation at Ser133 is not affected by S142A/S143A mutations. Left, pCREB133 is significantly reduced in tissue from CREBdn-S133A-injected mice versus mice injected with the control virus (p = 0.02, independent t test). Right, pCREB133 levels do not change in tissue from CREBdn-S142A/S143A-injected mice versus mice injected with the control virus (n.s. = not significant, p = 0.49, independent t test). The data in the gray box were previously published in J Neurosci (Pulimood et al., 2017). All error bars indicate standard error of the mean (SEM).

Activity-dependent Arc expression is blocked in the absence of CREB phosphorylation at Ser133 as well as at Ser142/143. A, Stimulus and virus infection conditions are as follows (from left): ACSF control media in cells infected with HSV-GFP control virus (n = 105 cells on 12 coverslips from 3 independent experiments), KCl-rich ACSF to depolarize cells infected with HSV-GFP control virus (n = 128 cells on 15 coverslips from 3 independent experiments), KCl-rich ACSF in cells infected with CREBdn-S133A (n = 51 cells on 20 coverslips from 3 independent experiments), and KCl-rich ACSF in cells infected with CREBdn-S142A/S143A (n = 63 cells on 15 coverslips from 3 independent experiments). Top panels show Arc staining in red and bottom panels show HSV infection in green. B, A cumulative distribution plot showing all analyzed cells in each condition clearly displays the activity-dependent increase in Arc expression that is blocked by the CREBdn viruses [one-way ANOVA, F_(3,333) = 53.1, p < 0.0001; Tukey’s post hoc test GFP(+KCl) vs all other groups p < 0.0001]. C, Representative case of Arc expression in mice injected with CREBdn-S142A/S143A or control GFP. Cyclophilin B (CyB) was used as a loading control. D, Quantification shows a significant reduction in Arc expression after blocked phosphorylation of CREB at Ser142/143 (t = 2.32, *p = 0.04; df = 8, t test for unequal variances). All error bars indicate standard error of the mean (SEM). OD = optical density.

VEP recordings: schematics of the experimental timeline and spike analysis. A, Electrode implantation and virus injection were done together at P26. After recovery from surgery, habituation followed by a pre-MD baseline recording was performed. The animal was then monocularly deprived by eyelid suture for a 7-d period, after which the deprived eye was reopened and a post-MD recording was conducted. VEP = visually evoked potential; HSV = herpes simplex virus; P = post-natal day. B, As the implanted mouse views the visual stimulus, electrical signals are transmitted through an amplifier, noise eliminator, and digitizer, and are recorded as the EEG signal shown in green. The amplitude of the VEP response (after synchronized averaging) is measured in microvolts (μV) from peak to trough as marked by the red lines.